https://www.alphaknockout.com Mouse Slit1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Slit1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Slit1 gene (NCBI Reference Sequence: NM_015748 ; Ensembl: ENSMUSG00000025020 ) is located on Mouse chromosome 19. 37 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 37 (Transcript: ENSMUST00000025993). Exon 6~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Slit1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-332I24 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a reporter allele exhibit normal interneuron numbers and morphology. Exon 6 starts from about 10.58% of the coding region. The knockout of Exon 6~8 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 992 bp, and the size of intron 8 for 3'-loxP site insertion: 1117 bp. The size of effective cKO region: ~1526 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 5 6 7 8 9 10 11 37 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Slit1 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8026bp) | A(20.57% 1651) | C(26.92% 2161) | T(26.56% 2132) | G(25.94% 2082) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 41651953 41654952 3000 browser details YourSeq 266 609 1053 3000 94.2% chr8 - 119806058 120000266 194209 browser details YourSeq 254 611 1051 3000 91.6% chr19 - 25706611 25707205 595 browser details YourSeq 236 609 1134 3000 89.6% chr10 - 9177593 9178118 526 browser details YourSeq 232 611 1115 3000 89.3% chr16 - 50118672 50119168 497 browser details YourSeq 232 609 1046 3000 92.2% chr5 + 76467499 76758541 291043 browser details YourSeq 225 611 1053 3000 88.4% chr4 + 111438230 111438798 569 browser details YourSeq 220 609 1046 3000 93.8% chr10 + 117438321 117438772 452 browser details YourSeq 213 615 1035 3000 90.8% chr6 - 127940320 127940797 478 browser details YourSeq 213 609 1034 3000 94.1% chr10 - 57300252 57300917 666 browser details YourSeq 208 683 1053 3000 89.3% chr10 - 45395658 45396070 413 browser details YourSeq 206 630 1043 3000 92.6% chr12 - 9443973 9444741 769 browser details YourSeq 205 609 1043 3000 89.2% chr4 + 111439232 111439664 433 browser details YourSeq 204 612 1051 3000 92.7% chr2 + 25761624 25762145 522 browser details YourSeq 198 615 1009 3000 93.2% chr1 + 78611640 78612187 548 browser details YourSeq 192 719 1053 3000 92.6% chr8 - 119999446 119999964 519 browser details YourSeq 191 609 1043 3000 91.4% chr4 + 111438712 111439158 447 browser details YourSeq 188 615 1053 3000 84.7% chr8 - 114424248 114424630 383 browser details YourSeq 188 611 1043 3000 92.3% chr4 + 111438370 111439576 1207 browser details YourSeq 186 609 1012 3000 94.9% chr11 + 81629423 81867531 238109 Note: The 3000 bp section upstream of Exon 6 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 41647427 41650426 3000 browser details YourSeq 24 2087 2110 3000 100.0% chr1 + 35840521 35840544 24 browser details YourSeq 24 2087 2110 3000 100.0% chr1 + 35840528 35840551 24 Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Slit1 slit guidance ligand 1 [ Mus musculus (house mouse) ] Gene ID: 20562, updated on 14-Aug-2019 Gene summary Official Symbol Slit1 provided by MGI Official Full Name slit guidance ligand 1 provided by MGI Primary source MGI:MGI:1315203 See related Ensembl:ENSMUSG00000025020 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Slil1; mKIAA0813 Expression Biased expression in whole brain E14.5 (RPKM 7.8), CNS E14 (RPKM 6.0) and 9 other tissues See more Orthologs human all Genomic context Location: 19; 19 C3 See Slit1 in Genome Data Viewer Exon count: 39 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (41597824..41747275, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (41674747..41818346, complement) Chromosome 19 - NC_000085.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Slit1 ENSMUSG00000025020 Description slit guidance ligand 1 [Source:MGI Symbol;Acc:MGI:1315203] Gene Synonyms Slil1 Location Chromosome 19: 41,600,257-41,743,665 reverse strand. GRCm38:CM001012.2 About this gene This gene has 5 transcripts (splice variants), 245 orthologues, 24 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Slit1-201 ENSMUST00000025993.9 5045 1531aa ENSMUSP00000025993.3 Protein coding CCDS29812 Q80TR4 TSL:1 GENCODE basic APPRIS P1 Slit1-203 ENSMUST00000169141.7 5673 1458aa ENSMUSP00000129034.1 Protein coding - E9Q2P0 TSL:5 GENCODE basic Slit1-202 ENSMUST00000166496.7 4556 1458aa ENSMUSP00000128381.1 Protein coding - E9QAW9 TSL:5 GENCODE basic Slit1-205 ENSMUST00000171586.1 1220 380aa ENSMUSP00000126139.1 Protein coding - E9Q117 CDS 3' incomplete TSL:5 Slit1-204 ENSMUST00000169416.1 516 No protein - Retained intron - - TSL:3 163.41 kb Forward strand 41.60Mb 41.65Mb 41.70Mb 41.75Mb Genes Gm22601-201 >misc RNA (Comprehensive set... Contigs < AC132303.3 < AC129182.4 Genes (Comprehensive set... < Slit1-203protein coding < Slit1-201protein coding < Slit1-202protein coding < Slit1-204retained intron < Slit1-205protein coding Regulatory Build 41.60Mb 41.65Mb 41.70Mb 41.75Mb Reverse strand 163.41 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000025993 < Slit1-201protein coding Reverse strand 143.41 kb ENSMUSP00000025... Low complexity (Seg) Cleavage site (Sign... Superfamily SSF52058 SSF57196 Concanavalin A-like lectin/glucanase domain superfamily SMART SM00364 EGF-like domain Cystine knot, C-terminal Leucine-rich repeat, typical subtype Laminin G domain SM00365 EGF-like calcium-binding domain Leucine-rich repeat N-terminal domain Follistatin-like, N-terminal Cysteine-rich flanking region, C-terminal Pfam Cysteine-rich flanking region, C-terminal EGF-like domain Laminin G domain Leucine-rich repeat N-terminal domain EGF-like, conserved site Leucine-rich repeat PROSITE profiles Leucine-rich repeat EGF-like domain Cystine knot, C-terminal Laminin G domain PROSITE patterns EGF-type aspartate/asparagine hydroxylation site EGF-like, conserved site Cystine knot, C-terminal EGF-like, conserved site EGF-like calcium-binding, conserved site PANTHER PTHR45836 PTHR45836:SF3 Gene3D Leucine-rich repeat domain superfamily 2.60.120.200 2.10.25.10 CDD cd00054 cd00110 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1531 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.